Plasmid DNA purification
MACHEREY-NAGEL – 08 / 2013, Rev. 01
8
Table 2: Information about antibiotics according to Maniatis*
Antibiotic
Stock solution
(concentration)
Storage
Working
concentration
Ampicillin
50 mg/mL in H
2
O
-20 °C
20–50 μg/mL
Carbenicillin
50 mg/mL in H
2
O
-20 °C
20–60 μg/mL
Chloramphenicol
34 mg/mL in EtOH
-20 °C
25–170 μg/mL
Kanamycin
10 mg/mL in H
2
O
-20 °C
10–50 μg/mL
Streptomycin
10 mg/mL in H
2
O
-20 °C
10–50 μg/mL
Tetracycline
5 mg/mL in EtOH
-20 °C
10–50 μg/mL
As rule of thumb use 5 mL
of a well grown LB culture as given in the kit specifications.
However, the culture volume can be increased if the cell culture grows very poorly or
has to be decreased if, e.g., very rich culture media were used. Refer to Table 3 to
choose the best culture volume according to the optical density at 600 nm (OD
600
).
Table 3: Recommended culture volumes for NucleoSpin
®
Plasmid EasyPure
OD
600
1
2
3
4
5
6
Culture volume
15 mL
8 mL
5 mL
4 mL
3 mL
2 mL
Note, if too much bacterial material is used, the lysis and precipitation steps become
inefficient causing decreased yield and plasmid quality! If more than the recommended
amount of cells shall be processed increase all lysis buffers proportionally.
2.4 Elution procedures
The elution buffer volume and method can be adapted to the subsequent downstream
application to achieve higher yield and / or concentration than the standard method
(recovery about 70–90 %):
•
Higher yield in general, especially for larger constructs: Heat elution buffer
to 70 °C, add 50–100 μL to the NucleoSpin
®
Plasmid EasyPure Column and
incubate at 70 °C for 2 min.
•
High yield: Perform two elution steps with the volume indicated in the individual
protocol. About 90–100 % of bound nucleic acids can be eluted.
* Maniatis T, Fritsch EF, Sambrook J:
Molecular cloning. A laboratory manual
, Cold Spring Harbor, Cold Spring,
New York 1982.
